Abstract: Pancreatic Ductal Adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related death in the USA. This dismal prognosis is due to late clinical presentation and resistance to therapy. The long term goal of our laboratory is to identify new, useful cancer-specific glycoprotein biomarkers of PDAC to facilitate early detection. Emerging evidence suggests that altered protein glycosylation may have utility as cancer biomarkers. We have demonstrated the over-expression of fucosyltransferase 3 (responsible for increased glycan fucosylation), as well as the decreased expression of two isoforms of mannosidase (responsible for less frequent removal of mannose from high mannose core structures in glycans, and as a result, increased branching of the high mannose cores). These enzymes may play a role in the appearance of altered N-linked and O-linked glycan structures found in cancer. PDAC tumors have alterations in N-linked and O-linked glycan moieties on specific glycoproteins in comparison to the same proteins found in normal tissues. Our first aim will define cancer-specific changes in the N-linked and O-linked glycoprotein content of PDAC, chronic pancreatitis and normal pancreata, using liquid-based protein separations to look for protein expression differences. Glycoproteins that exhibit expression differences will be identified by MS/MS. Identified proteins with significant and meaningful differences in expression will be evaluated to assess tumor tissue specificity by IHC. In our second aim we seek to define alterations in N-linked and O-linked glycan structure using high-density glycoprotein arrays. High-density protein microarrays will be constructed using all of the liquid-based fractions generated in our first aim. These arrays will be utilized to study N-linked and O-linked glycan structure as a function of cancer using the reactivity of tumor glycoproteins to fluorescently labeled lectins as a probe, as measured by differences in the intensity of the fractions arrayed from the individual samples. Glycoproteins showing statistically significant and biologically meaningful differential expression between PDAC and both normal and chronic pancreatitis will be identified. We will use multivariate analysis techniques, including hierarchical clustering (HC) and principal components analysis (PCA), to identify multivariate predictors of PDAC. Identification of glycoproteins showing altered N-linked or O-linked glycan structure will be performed by MS/MS, and the tumor cell specificity verified by IHC and/or by quantitative IHC. In our third aim we seek to identify specific glycan structure alterations of PDAC tissue glycoproteins. We will isolate glycans and structurally analyze the glycans in a (QIT)-TOF MALDI mass spectrometer where MS2, MS3 and MS4 can be used for detailed structural analysis. In our fourth aim we seek to determine whether the candidate glycoprotein biomarkers can be found in plasma from patients with PDAC as compared to normal age/sex matched control plasma. These over-expressed or altered tumor glycoproteins may have utility as biomarkers for early detection of PDAC.